Project summary Peroxisomes carry out many key functions related to ROS and lipid metabolism, including oxidation of very long chain fatty acids and synthesis of ether lipids. As highly plastic organelles, peroxisomes can modify their size, morphology, abundance, and function, depending on external stimuli. Our preliminary studies suggest that peroxisomal biogenesis increases in adipose tissue in response to cold exposure, in a manner dependent on the thermogenic transcription factor PRDM16, consistent with the possibility that peroxisomes are involved in thermogenesis. Using a new mouse model of adipose-specific peroxisome deficiency, we discovered that peroxisomes are critical for brown fat-mediated thermogenesis and that the loss of peroxisomes in adipose tissue is associated with decreased energy expenditure and increased adiposity. By pursuing the molecular mechanism through which peroxisomes regulate adipose tissue thermogenesis, our preliminary studies implicate peroxisomes in mitochondrial division. As dynamic organelles, mitochondria undergo repeated cycles of fission and fusion. Combined with the cold-induced stimulation of lipolysis, activation of mitochondrial fission is thought to serve as a critical physiological regulator of energy expenditure and thermogenic function of brown fat. Our studies suggest that the loss of peroxisomes impairs cold-induced mitochondrial fission and decreases mitochondrial DNA content in BAT. These phenotypes do not appear to be related to impaired ability of peroxisomes to oxidize very long chain fatty acids or through an altered redox state in brown adipocytes in the absence of peroxisomes. Instead, our results implicate the ability of peroxisomes to synthesize a type of ether lipids called plasmalogens in the mechanism. Plasmalogens are present in the mitochondrial membrane and inhibition of their synthesis in brown adipocytes mimics the effect of blocking peroxisomal biogenesis on mitochondrial morphology. These data lead us to hypothesize that peroxisomes regulate adipose tissue thermogenesis by channeling plasmalogens to mitochondria to mediate mitochondrial fission. We propose two specific aims to test this hypothesis. In Aim 1, we will elucidate the molecular mechanism through which peroxisomes regulate mitochondrial dynamics and adipose tissue thermogenesis. The second Aim will study the role of peroxisomal lipid synthesis in mitochondrial function, thermogenesis, and metabolic homeostasis using mice with adipose- specific inactivation of plasmalogen synthesis. Together, this work will provide a significant mechanistic insight into the role of peroxisomes in adipose tissue thermogenesis. Understanding how peroxisomes regulate mitochondrial fission could lead to novel strategies to exploit the thermogenic function of brown fat for treatment of obesity and diabetes.